Long-term in vitro culture of Plasmodium vivax isolates from Madagascar maintained in Saimiri boliviensis blood.

BACKGROUND: Plasmodium vivax is the most prevalent human malaria parasite and is likely to increase proportionally as malaria control efforts more rapidly impact the prevalence of Plasmodium falciparum. Despite the prominence of P. vivax as a major human pathogen, vivax malaria qualifies as a neglected and under-studied tropical disease. Significant challenges bringing P. vivax into the laboratory, particularly the capacity for long-term propagation of well-characterized strains, have limited the study of this parasite's red blood cell (RBC) invasion mechanism, blood-stage development, gene expression, and genetic manipulation. METHODS AND RESULTS: Patient isolates of P. vivax have been collected and cryopreserved in the rural community of Ampasimpotsy, located in the Tsiroanomandidy Health District of Madagascar. Periodic, monthly overland transport of these cryopreserved isolates to the country's National Malaria Control Programme laboratory in Antananarivo preceded onward sample transfer to laboratories at Case Western Reserve University, USA. There, the P. vivax isolates have been cultured through propagation in the RBCs of Saimiri boliviensis. For the four patient isolates studied to-date, the median time interval between sample collection and in vitro culture has been 454 days (range 166-961 days). The median time in culture, continually documented by light microscopy, has been 159 days; isolate AMP2014.01 was continuously propagated for 233 days. Further studies show that the P. vivax parasites propagated in Saimiri RBCs retain their ability to invade human RBCs, and can be cryopreserved, thawed and successfully returned to productive in vitro culture. CONCLUSIONS/SIGNIFICANCE: Long-term culture of P. vivax is possible in the RBCs of Saimiri boliviensis. These studies provide an alternative to propagation of P. vivax in live animals that are becoming more restricted. In vitro culture of P. vivax in Saimiri RBCs provides an opening to stabilize patient isolates, which would serve as precious resources to apply new strategies for investigating the molecular and cellular biology of this important malaria parasite.

Pattern of maternal circulating CRH in laboratory-housed squirrel and owl monkeys.

The anthropoid primate placenta appears to be unique in producing corticotropin-releasing hormone (CRH). Placental CRH is involved in an endocrine circuit key to the production of estrogens during pregnancy. CRH induces cortisol production by the maternal and fetal adrenal glands, leading to further placental CRH production. CRH also stimulates the fetal adrenal glands to produce dehydroepiandrostendione sulfate (DHEAS), which the placenta converts into estrogens. There are at least two patterns of maternal circulating CRH across gestation among anthropoids. Monkeys examined to date (Papio and Callithrix) have an early-to-mid gestational peak of circulating CRH, followed by a steady decline to a plateau level, with a possible rise near parturition. In contrast, humans and great apes have an exponential rise in circulating CRH peaking at parturition. To further document and compare patterns of maternal circulating CRH in anthropoid primates, we collected monthly blood samples from 14 squirrel monkeys (Saimiri boliviensis) and ten owl monkeys (Aotus nancymaae) during pregnancy. CRH immunoreactivity was measured from extracted plasma by using solid-phase radioimmunoassay. Both squirrel and owl monkeys displayed a mid-gestational peak in circulating CRH: days 45-65 of the 152-day gestation for squirrel monkeys (mean±SEM CRH=2,694±276 pg/ml) and days 60-80 of the 133-day gestation for owl monkeys (9,871±974 pg/ml). In squirrel monkeys, circulating CRH declined to 36% of mean peak value by 2 weeks before parturition and then appeared to increase; the best model for circulating CRH over gestation in squirrel monkeys was a cubic function, similar to previous results for baboons and marmosets. In owl monkeys, circulating CRH appeared to reach plateau with no subsequent significant decline approaching parturition, although a cubic function was the best fit. This study provides additional evidence for a mid-gestational peak of maternal circulating CRH in ancestral anthropoids that has been lost in the hominoid lineage.

Ovarian stimulation of squirrel monkeys (Saimiri boliviensis boliviensis) using pregnant mare serum gonadotropin.

The application of assisted reproductive technologies (ART) to nonhuman primates has created opportunities for improving reproductive management in breeding colonies, and for creation of new animal models by genetic modification. One impediment to the application of ART in Saimiri spp. has been the lack of an effective gonadotropin preparation for ovarian stimulation. Pregnant mare serum gonadotropin (PMSG) is inexpensive and readily available, but its repeated use in rhesus monkeys has been associated with induction of a refractory state. We have compared PMSG to recombinant human follicle stimulating hormone (rhFSH) for controlled ovarian stimulation in Bolivian squirrel monkeys. Groups of mature squirrel monkeys received rhFSH (75 IU daily) or PMSG (250 IU twice daily) by subcutaneous injection for 4 d during the breeding season (November to January) or nonbreeding season (March to September). Serum estradiol (E2) was measured daily. Follicular growth was monitored by abdominal ultrasound. During the breeding season, PMSG induced a higher E2 response than did rhFSH, with mean E2 levels being significantly higher within 3 d of stimulation. Superior follicular development in PMSG animals was confirmed by abdominal ultrasonography. During the nonbreeding season, PMSG elicited a similar increase in serum E2 levels despite the fact that basal serum E2 is typically low during the nonbreeding season. Repeated use of PMSG (< or = 3 cycles of administration) produced no attenuation of the E2 response. We conclude that PMSG is highly effective for repeated cycles of controlled ovulation stimulation in the squirrel monkey.

Histocytological specificities of adrenal cortex in the New World Monkeys, Aotus lemurinus and Saimiri boliviensis.

The New World monkey Aotus spp. (night monkeys) are expected for use of valuable experimental animal with the close species of Saimiri spp. (squirrel monkeys). Saimiri is known to show spontaneous hypercortisolemia, although few reports in Aotus. We compared basic states of blood steroid hormones and histological structure of the adrenal glands in two monkeys. Serum cortisol and ACTH levels were statistically lower in Aotus than Saimiri. Conversely, Aotus adrenocortical area showed significant enlargement, especially at the zona fasciculata. Electron microscopic observation at Aotus fasciculata cells revealed notable accumulation of large lipid droplets and irregular shapes of the mitochondrial cristae. These results suggest potential differences in cellular activities for steroidogenesis between Aotus and Saimiri and experimental usefulness in adrenocortical physiology and pathological models.

Triacylglycerol and very low density lipoprotein secretion into plasma of squirrel monkeys.

We determined the effects of varying the types and level of dietary fat and cholesterol on the increase in plasma total triacylglycerol concentrations after injection of Triton WR-1339, an inhibitor of lipoprotein lipase, into monkeys that had been subjected to an overnight fast. The monkeys that had been treated with Triton WR-1339 were then given a test meal by intragastric intubation. Dietary cholesterol, high levels of fat and saturated fat in the habitual diet reduced the rate of release of triacylglycerol to plasma in the fasted monkey. We also determined the changes in protein and lipid concentrations of the different lipoprotein fractions. The injection of Triton WR-1339 resulted in a linear increase with time in the concentration of protein and triacylglycerol in the very low density (chylomicron-free and d less than 1.006) lipoproteins, but there was an increase in the ratio of traicylglycerol to protein in that fraction. Most of the increase (96%) in very low density protein was in the B protein. Regardless of the habitual diet, a test meal accentuated the rate of triacylglycerol appearance in whole plasma and in the very low density lipoproteins of Triton WR-1339-treated monkeys, and the rate of increase of the protein component after feeding was slightly higher. Thus the administration of a meal to the fasted Triton WR-1339-treated squirrel monkey further increased the proportion of triacylglycerol in very low density lipoproteins. Although dietary cholesterol and saturated fat in the habitual diet depressed the rate of increase in very low density triacylglycerol during fasting, the rate of protein synthesis was not significantly affected. After administration of a test meal the rates of increase in triacylglycerol and protein in the very low density lipoproteins were similar for monkeys from the different diet groups. Triton WR-1339 administration caused a slight and progressive increase in the intermediate density (d 1.006-1.019) lipoproteins and a marked and progressive decrease in the low density (d 1.019-1.063) lipoproteins. There was an immediate (by 5 min) drop of 70% or more in high density (d 1.063-1.21) lipoprotein protein, but the lipids except triacylglycerol remained unchanged. There was a decrease in both the A (the major fraction) and C proteins. The rates of very low density B protein secretion were comparable to the rates of low density lipoprotein catabolism that had been previously demonstrated for this species.

Metabolism of lipoproteins in nonhuman primates. Studies on the origin of low density lipoprotein apoprotein in the plasma of the squirrel monkey.

The plasma of squirrel monkeys contains extremely low levels of very low density lipoproteins. The delipidated apoproteins from the different lipoprotein density classes of this species show a heterogeneity similar to that of man and the rat. The biosynthesis of the apoproteins of squirrel monkey lipoproteins was studied in fasted normal and Triton WR1339-treated animals. After intravenous injection of [3-H] leucine, maximal labeling of very low density lipoproteins occurred after 1 h, intermediate density lipoproteins (d 1.006--1.019) in 2 h, and low density lipoproteins after 3 h. At all times, however, low density lipoproteins had the greatest percentage of radioactivity. Polyacrylamide gel electrophoresis revealed that the apoprotein B moiety of very low density and intermediate density lipoproteins contained 62% and 81% of the total radioactivity in these lipoproteins whereas the fast-migrating peptides were minimally labeled. In monkeys injected with Triton WR1339, 70--80% of the radioactivity incorporated into d smaller than 1.063 lipoproteins was in very low density lipoproteins with only 10--15% in intermediate and low density lipoproteins. After injection of 3-H-labeled very low density lipoproteins and [14-C] leucine into Triton-treated monkeys, catabolism of 3-H-labeled very low density lipoprotein to intermediate and low density lipoproteins was small and was significantly less than corresponding values for the incorporation of [14-C] leucine. Thus, breakdown of very low density lipoproteins could not account for all the labeled apoprotein B present in the intermediate and low density lipoprotein fractions. The results indicate that most, but not all, of the newly synthesized apoprotein B enters plasma in very low density lipoproteins and that the low concentrations of this lipoprotein in squirrel monkey plasma are a consequence of its rapid turnover.

Blood typing in Saimiri sciureus monkeys: influence of anti-red blood cell alloantibodies on Plasmodium falciparum parasitaemia in vivo.

The Saimiri sciureus monkey is a well-established host for experimental studies with human malaria parasites. During the course of iterative inoculations with Plasmodium falciparum parasitised red blood cells (RBC), anti-RBC alloantibodies were detected in the sera of two of eight Saimiri monkeys. These anti-RBC antibodies were further used to investigate RBC phenotypes in 35 colony-reared Saimiri monkeys by flow cytometry. Three RBC phenotypes (named I-III) were observed. Their distribution was I (86%), II (11%) and III (3%). Using the Palo Alto FUP-2 strain, a variant P. falciparum line insensitive to hyperimmune serum and the passive transfer of anti-RBC alloantibodies, a dramatic drop in parasite growth was documented in an incompatible monkey.

Serum leptin in nonpregnant and pregnant women and in old and new world nonhuman primates.

Leptin is a hormone that is produced during mammalian pregnancy in the placental trophoblast and other tissues, including! fetal and maternal adipocytes. Synthesis of the polypeptide and the presence of its specific receptors throughout the human maternal fetoplacental unit suggest direct effects on conceptus growth and development. However, both the physiologic roles of leptin and the mechanisms regulating leptin synthesis in human pregnancy differ from those in laboratory and domestic species, necessitating the development of non-human primate research models. Therefore, we compared serum leptin concentrations in nonpregnant and pregnant women with those in both old world nonhuman primates (i.e., baboon, rhesus monkey, cynomolgus monkey) and new world nonhuman primates (i.e., squirrel monkey, titi monkey). As expected, maternal leptin levels were elevated in human and baboon pregnancies (P < 0.05 and P < 0.001, respectively). Levels in both species of old world monkeys were also greatly enhanced (P < 0.001). Although maternal serum concentrations were slightly elevated compared to nonpregnant levels in both species of new world monkeys, overall concentrations were dramatically lower than for either old world primates or humans. Results provide comparisons of serum leptin concentrations in pregnant and nonpregnant humans and baboons with those in both old and new world monkeys and further characterize these nonhuman primates as models for the investigation of leptin dynamics in pregnancy.

Rapid cortisol and corticosteroid-binding globulin responses during pregnancy and after estrogen administration in the squirrel monkey.

Plasma cortisol and corticosteroid-binding globulin (CBG) levels were assessed in pregnant squirrel monkeys and in intact and castrated males after estrogen administration. Pregnant females showed a rapid and dramatic rise in cortisol and CBG during the first 8 weeks after conception. Estrogen treatment also caused marked elevations in cortisol and CBG. Cortisol levels increased significantly by 24 h after estrogen injection and remained elevated for 6 weeks of treatment, but a relatively greater rise in CBG resulted in a higher CBG/cortisol ratio. The data support prior research indicating that estrogen can simultaneously stimulate adrenal output and the compensatory binding of circulating cortisol by increased CBG synthesis. In addition, it appears that even in the absence of exogenous treatment, the pituitary-adrenal axis of male squirrel monkeys is stimulated by estrogen derived either from the testes or by the peripheral conversion of testosterone to estrogen.

Manipulating blood T cells and B cells from squirrel monkeys: some technical considerations.

The squirrel monkey Saimiri sciureus is an experimental host for a range of human pathogens, and for the assessment of vaccine candidate antigens and vaccine strategies. This experimental host is thus particularly suitable for the follow-up of humoral responses. To understand some of the mechanisms that underlie the defense against experimental pathogens, there is a need of basic knowledge on cellular immune effectors also. The authors report here their experience in characterizing squirrel monkey blood T and B lymphocytes, and in studying in vitro induced activation and proliferation of T and B cells. Particular emphasis is given to the in vitro differentiation of squirrel monkey B cells into immunoglobulin secreting cells, with respect to Plasmodium falciparum antigens.

In vitro fibrinolytic activity of recombinant tissue-type plasminogen activator in the plasma of various primate species.

The ratio of fibrinolytic to fibrinogenolytic effect of recombinant human tissue-type plasminogen activator was investigated at several concentrations in an in vitro system consisting of 125I-fibrin labeled autologous plasma clots immersed in the plasma of several different primate species. A concentration-dependent fibrinolysis was obtained in each case; however, the degree of fibrinolysis differed markedly from one primate species to the other. At a concentration of 30 IU/ml (300 ng/ml) of recombinant tissue-type plasminogen activator nearly complete thrombolysis was achieved within 4 hr in human plasma, in the chimpanzee, in the cynomolgus macaque, in the bonnet macaque and in the rhesus macaque. The common baboon, the olive baboon and the squirrel monkey were rather resistant to thrombolysis. In all species the thrombolysis achieved was not associated with fibrinogenolysis nor with significant decreases in plasminogen or alpha 2-antiplasmin.

Cortisol responses under different housing conditions in female squirrel monkeys.

Adult female squirrel monkeys were housed in a group, in pairs or individually. Plasma levels of cortisol obtained under basal and stress conditions once weekly for four weeks were significantly lower in pair-housed females than in those living in a social group or individually. The increment in cortisol levels after stress (induced by handling and ether anesthesia) also was smaller in females housed in pairs. The cortisol values of the pair-housed females were positively correlated with those of their partners. Basal cortisol levels in the group-living females showed a significant rank-order correlation with dominance status. This indicated that social interactions in group-living animals can influence cortisol levels in a complex manner, either increasing or decreasing them. The relatively lower pituitary--adrenal activation when a single partner was present also indicated that the social environment can affect an individual's general level of arousal and subsequently alter the response to stressful stimuli.

Plasma butyrylcholinesterase activity and cocaine half-life differ significantly in rhesus and squirrel monkeys.

In vitro studies have implicated butyrylcholinesterase (BChE, E.C.3.1.1.8) as the major enzyme for metabolizing cocaine in humans, but little is known about endogenous BChE activity in monkeys and other animals often used in preclinical studies of cocaine. We compared BChE activity in 18 rhesus and 11 squirrel monkeys, using the colorimetric method of Ellman with butyrylthiocholine as substrate, and in vitro cocaine half-life in pooled plasma samples measuring cocaine concentrations over 60 minutes by GC-MS. Rhesus monkeys had a significantly higher plasma BChE activity than squirrel monkeys (8.2 +/- 0.5 U/L vs. 2.8 +/- 0.5 U/L), and a three-fold shorter in vitro cocaine half-life (20.1 min vs. 60.2 min). BChE activity in rhesus monkeys was comparable to the activity reported in humans. There was no significant influence of age, weight, or prior cocaine exposure. These results indicate that BChE level can vary between species of non-human primates, a factor that should be taken into account when studying drugs such as cocaine which are metabolized by BChE.

Behavioral and endocrine consequences of heterosexual pair formation in squirrel monkeys.

The South American squirrel monkey (Saimiri sciureus) typically lives in large social groups containing several individuals of all age/sex categories. When living in established heterosexual pairs, reproduction in this seasonally breeding primate is poor. We attempted to induce breeding activity in pair-housed monkeys by forming new heterosexual pairs just prior to the breeding season. Breeding readiness, as reflected in behavior and gonadal hormones, was induced in males, but not in females. Males also showed persistent increases in cortisol levels following formation of new heterosexual pairs; females did not. The results indicate that social stimulation provided by a single novel female is sufficient to enhance breeding readiness in male squirrel monkeys. Females, on the other hand, are apparently unresponsive to a single male whether novel or familiar; this may account for the poor reproductive success in squirrel monkeys housed in heterosexual pairs.

High plasma steroid levels in the squirrel monkey: deficient receptors or metabolism?

Many New World primates such as the squirrel monkey have extraordinarily high plasma steroid hormone levels as compared to humans and Old World primates. To clarify the mechanism(s) underlying this apparent steroid resistance, glucocorticoid and androgen binding to putative receptors in genital skin fibroblasts from several species was investigated. Differences in either affinity and/or number of binding sites were found but these were small compared to the very large differences in total or free plasma steroid concentrations between Old and New World primate species. In contrast, when the ability of fibroblasts to metabolize testosterone was compared, squirrel monkey cells were devoid of 5 alpha-reductase activity which was readily demonstrated in human cells. Together with other data indicating that squirrel monkeys excrete little if any 5 alpha- or 5 beta-reduced urinary steroid metabolites, these results suggest that inefficient metabolism rather than receptor binding abnormalities may account for the elevated plasma hormone levels in the squirrel monkey.

Phylogenetic variability of serum lipids and lipoproteins in non-human primates fed diets with different contents of dietary cholesterol.

The response of serum lipids and lipoproteins to different levels of dietary cholesterol (0.05% to 1.5% w/w) was measured in six nonhuman primate species. Relative response of serum cholesterol in different species, measured in terms of response, index, varied with dietary cholesterol concentration. The overall response for the different diets allowed ranking of the species as follows: Squirrel is greater than green is greater then spider is approximately thesus is approximately patas is greater than chimpanzee The serum cholesterol response was reflected not only in an increase in beta + pre-beta-lipoprotein cholesterol but also in alpha-lipoprotein cholesterol, with significant differences among species in the amount of cholesterol transported in the lipoprotein classes.

Erythrocyte characteristics in vitamin E-responsive anemia of the owl monkey (Aotus trivirgatus).

To characterize the vitamin E-responsive anemia occurring in owl monkeys (Aotus trivirgatus), osmotic fragility, and H2O2-induced and time-dependent hemolysis, as well as RBC lipid peroxidation, were compared in anemic and nonanemic owl monkeys. Whereas vitamin E serves as a lipid-soluble antioxidant, the glutathione peroxidase system functions in the water-soluble phase of the cell. Thus, activity of glutathione peroxidase, glutathione reductase, and glucose-6-phosphate dehydrogenase, as well as reduced glutathione concentrations in owl monkeys' RBC, were compared with those of rhesus macaques and cebus and squirrel monkeys fed the same diet and maintained under the same management scheme. Osmotic fragility did not differ between anemic and nonanemic owl monkeys. The H2O2-induced and time-dependent hemolysis was approximately 10-fold greater among anemia owl monkeys than among their nonanemic counterparts, and lipid peroxidation values tended to be higher in the anemic monkeys. Owl monkeys, as a species and independent of anemia, exhibited higher RBC peroxidation than did 2 other New World species, cebus and squirrel monkeys. The glutathione peroxidase system was not depressed in owl monkey RBC. The only observed difference in this system was in the glucose-6-phosphate dehydrogenase activity, which was 3- to 6-fold higher in the owl monkey than in the other species, indicating an increased activity of the peroxidase system. Thus, a defect in the glutathione peroxidase system could not be identified.

Specific gravity of whole blood in cynomolgus monkeys (Macaca fascicularis), squirrel monkeys (Saimiri sciureus), and tamarins (Saguinus labiatus) and total blood volume in cynomolgus monkeys.

Blood collection is a common laboratory procedure in animal experiments. The purpose of this study is to establish baseline data for two essential hematologic parameters, total blood volume (TBV) and specific gravity of blood (SGB), of nonhuman primates. The SGB was determined by dropping samples of whole blood into cupric sulfate solution. The values for the mean SGB +/- 1 standard deviation are: cynomolgus monkeys, 1.0526 +/- 0.0019 [males (n = 39), 1.0531 +/- 0.0017; females (n = 48), 1.0522 +/- 0.001]; squirrel monkeys, 1.0555 +/- 0.0037 [males (n = 56), 1.0581 +/- 0.0027; females (n = 76), 1.0536 +/- 0.0032]; and tamarins, 1.0582 +/- 0.0020 [males (n = 13), 1.0582 +/- 0.0023; females (n = 17), 1.0581 +/- 0.0018]. To determine the TBV, blood was collected in tubes containing 1.5 mg EDTA after intravenous injection of Evans Blue solution. The TBV was obtained after correcting for the hematocrit and the dilution factor of the Evans Blue solution. The formulae were established to estimate TBV by referring to body weight (BW). There was no significance between TBV and BW in male monkeys weighing more than 6 kg.

Non-ABO blood group systems phenotyping in non-human primates for blood banking laboratory and xenotransplantation.

Some biomedical research procedures, such as organ xenotransplantation, usually require intensive hemotherapy. Knowledge of the whole phenotype of blood donor and graft could be useful in the field of xenotransplantation. Human and simian-type categories of blood groups have been established and they can be tested by standard methods used for human blood grouping. The aim of this work was to study the incidence of non-ABO blood group systems in different species of non-human primates, which are employed in biomedical research. The phenotype of Rh, Lewis, Kidd, Kell, MNSs, Lutheran, P and Duffy antigens was investigated in olive baboon (n = 48), chacma baboon (n = 9), Guinea baboon (n = 14), Rhesus macaque (n = 38) and squirrel monkey (n = 30) by using commercial microtyping cards. Kell, Lutheran, Kidd and Duffy antigens have been detected in all species, Rh in squirrel monkey, MNSs in rhesus macaque and squirrel monkey, and Lewis in baboon and rhesus macaque. There were differences in frequency and haemagglutination scores between species regardless of their gender and age. The main differences were found in squirrel monkey when compared with baboons and macaques. This typing system provides a tool to assess the presence of antigens in animals used for experimental procedures, such as xenotransplantation and xenotransfusion.